Article Figures & Data
Figures
- Fig. 1.
Impact of RGS2 mutations on AT1R-mediated intracellular calcium release. (A) RGS2 mutation map. Needle plot showing the two-dimensional positions of 16 missense mutations on the human RGS2 protein. The plot was visualized by inputting amino acid changes of RGS2 protein to MutationMapper tool (cBioPortal). CHO cells transiently expressing AT1R receptor with or without RGS2 WT or RGS2 mutants were used for measurement of intracellular Ca2+. (B and C) Representative fluorescence traces (angiotensin II, 1 μM) from cells expressing AT1R alone (black), cells coexpressing AT1R and RGS2 WT, K18N (normal function), or D40Y (decreased function). (D and E) Concentration–response curves. K18N inhibits AT1R-mediated Ca2+ as equally as RGS2 WT while D40Y showed reduced inhibition as compared with WT. (F) Bar graph of maximal percentage inhibition mediated by RGS2 WT or mutants. Reduced function mutants are shown in dark gray (Q2L, R188H, R44H, D40Y) and mutants that did not show a significant difference from WT (uncorrected P > 0.05) are shown in light gray. ****P < 0.0001 compared with AT1R + control, #P < 0.05 compared with AT1R + RGS2 WT (n = 6–8, Student t test).
- Fig. 2.
Protein expression of RGS2 WT and mutants. CHO cells were transiently transfected with the V5-tagged (RGS2-V5) constructs; RGS2 protein levels were analyzed by anti-V5 Western blot. (A) Representative Western blot of 10 independent experiments showing results with total cell lysates of CHO cells expressing RGS2 WT and RGS2 mutant proteins. (B) Quantification of band intensities is normalized to the RGS2 WT protein level. Data are presented as mean ± S.E.M. *P < 0.05 (one-way analysis of variance with Bonferroni post-test).
- Fig. 3.
Effects of RGS2 missense mutations on RGS2-GFP localization. (A–C) When overexpressed in HEK293 cells, RGS2-GFP is localized to the nucleus. (D and H) Unlike the other mutants, the R188H-GFP mutant showed punctate intracellular localization. (E) Coexpression of WT-RGS2-GFP with constitutively active Gαq results in translocation of the RGS2-GFP to the plasma membrane. (F and G) This translocation is impaired with D40Y and R44H; these two mutants remained in the nucleus when coexpressed with Gαq Q209L. (I) Representative line scans across cells expressing WT, D40Y, R44H RGS2-GFP without Gαq. (J) Representative line scans across cells expressing WT + Gαq, D40Y + Gαq or R44H + Gαq. (K) Localization of RGS2 in at least 100 cells determined by a blinded observer shows impaired membrane localization of D40Y and R44H. ****P < 0.0001, one-way analysis of variance with Bonferroni post-test (scale bar: 20 μm).
- Fig. 4.
Impaired Gαq binding by the R188H mutant. (A) Binding affinities of RGS2 proteins with Gαq measured in a bead-based flow cytometry competition binding assay. As described in methods, Gαq was immobilized on beads, and mutant proteins were used to compete for binding of AF532 labeled WT-RGS2. The R188H mutant exhibited a reduction in Gαq binding affinity compared with RGS2 WT protein, demonstrated by the rightward shift in the competition binding curve. The Q78H, A99G, and Q196R had comparable binding affinities. (B) Thermoshift analysis of the RGS spanning domains from RGS2 WT, R188H, and Q196R. The bar graph shows the melting temperature of these proteins as mean ± S.E.M. from three independent experiments. *P < 0.05; ****P < 0.0001 (one-way analysis of variance with Bonferroni post-test). (C and D) Proposed structural mechanism of the impaired Gαq binding and thermal instability of the R188H mutant RGS2. Structure of the RGS2 domain (violet) – Gαq complex (cyan) (Nance et al., 2013) is shown. (C) The WT arginine at position 188 forms salt bridges with the glutamate residue at position 104. (D) Histidine substitution at position 188 does not favor salt bridge formation.
Tables
- TABLE 1
Missense mutations in the coding region of RGS2 used in this study
ExAC denotes the Exome Aggregation Consortium (http://exac.broadinstitute.org).
Mutation ID Missense Variant Allele Count (ExAC) Codon Change Reference NA Q2La 2/121304 CAA > CTA Yang et al. (2005); Lek et al. (2016) rs141030117 Q2R 65/121312 CAA > CGA Yang et al. (2005); Lek et al. (2016) rs145125159 S3G 32/121316 AGT > AGC Yang et al. (2005); Lek et al. (2016) rs142499684 A4V 4/121338 GCT > GTT Lek et al. (2016) rs193051407 M5V 82/121346 ATG > GTG Yang et al. (2005); Lek et al. (2016) rs74466425 K18N 34/121370 AAG > AAC Lek et al. (2016) rs148489044 G23D 66/121358 GGC > GAC Lek et al. (2016) rs201233692 D40Y 62/118360 GAT > TAT Lek et al. (2016) rs200339834 R44Ha 12/119308 CGT > CAT Yang et al. (2005); Lek et al. (2016) rs80221024 Q50K 103/119910 CAA > AAA Lek et al. (2016) rs140811638 P55L 6/120456 CCT > CTT Lek et al. (2016) NA Q78H 3/2055 CAG > CAC Yang et al. (2005) rs139237239 A99G 2/121332 GCT > GGT Lek et al. (2016) rs146862218 I110V 53/121334 ATT > GTT Lek et al. (2016) rs369752935 R188H 4/121388 CGT > CAT Lek et al. (2016) rs112707798 Q196R 18/121352 CAG > CGG Lek et al. (2016) NA, not applicable.
↵a RGS2 mutations were found in disease cohort (Yang et al., 2005).
Data Supplement
- Supplemental Data -
Supplemental Table 1 - Effect of RGS2 WT and mutant on AT1 receptor-mediated intracellular calcium release demonstrated by Max and EC50 derived AngII concentration response curves (* p< 0.05, *** p<0.001, compared to Ctrl; # p<0.05 compared to RGS2 WT)
Supplemental Figure 1 - MBP-RGS2 protein purification
Supplemental Figure 2 - Increasing amount of RGS2-V5 increases suppression of AT1R-mediated Ca2+ mobilization
Supplemental Figure 3 - Co-expression with RGS2 WT or mutants does not change HA-AT1R protein expression
Supplemental Figure 4 - Protein half-live of the Q2L and R188H
Supplemental Figure 5 - Initial fluorescent intensity of RGS2 WT, R188H and Q196R in DSF analysis
Supplemental Figure 6 - Effect of proteasomal inhibition on expression of R188H
SUPPLEMENTAL REFERENCES
- Supplemental Data -
In this issue
Altered Functions of RGS2 Missense Mutations
